Spatial genomic tools to interrogate T cell clonotypes, tumor clones and the microenvironment

点击翻译按钮获取中文摘要

The contemporary clinical successes of immunotherapies have highlighted the key role of tumor-infiltrating cells in mediating anti-tumor immunity and have generally associated the presence of T cells within tumors with therapeutic response. However, until now, a systematic approach for evaluating how T cell state, their clonal identity and localization are related has not been possible. In recent years, the Wu lab has achieved several notable technical advances, including generation of a best-in-class HLA class I epitope predictor (HLAthena); a highly robust targeted plate-based method for single-cell TCR sequencing (rhTCRseq); and a means to parallelize the cloning of hundreds of TCRs such that they can be interrogated to definitively link a TCR with its antigen specificity. Because dynamic interactions between tumor-reactive T cells and malignant clones occur within the defined spatial ordering of tissue, our valuable new insights motivate us to investigate how the spatial organization of tumor-specific T cells relates to in situ positioning of tumor clones. We hypothesize that antigen specificity, which drives the interactions between T cells and tumor cells, impacts the distinct regional localization of T cells within the tumor microenvironment at baseline and in the context of therapy; conversely, that knowledge of spatial localization identifies T cell clones specific for distinct antigen types. Slide-seq technology, created by the Chen lab, provides a tractable and exciting path to investigate this hypothesis by implementing a scalable approach to undertake in-depth analyses of informative human and murine tumor tissues. By expanding the capabilities of this unbiased cellular resolution spatial capture method, we aim to gain tissue level understanding of how the abundance and functional state of T cell clones and their spatial orientation within the tumor microenvironment are linked. In particular, the study will address the spatial organization of T cell clones and tumor subclones in human and mouse tumors. Our technology goals will be to increase the efficiency of transcript capture of the technology, extend the capability to include robust detection of tumor mutations, and to develop a suite of analytic tools to integrate in a multi model fashion the transcript, DNA-level and TCR levels of information (Aims 1-2). Focusing on RCC tumors, we will evaluate the cell-cell localization patterns of T cell clones in relation to tumor subclones and stromal cells through integrated spatial analyses of TCR and DNA Slide-seq data (Aim 2). Finally, we will evaluate the impact of antigen specificity (definitively assessed by robust TCR reconstruction and interrogation methods established in our lab) on T cell phenotype and localization using TCR and DNA Slide-seq integrated spatial analyses at baseline and following immune checkpoint blockade and neoantigen vaccine. (Aim 3) Altogether, we will develop and test these tools to understand spatially localized cellular networks which drive immune response, and T-cell receptor relationships with the tumor microenvironment. The completion of our work will yield a comprehensive toolset to enable a molecular, cellular and histological understanding of the tumor immune response.